Method Development and Validation for Determination of Febuxostat from Spiked Human Plasma Using RP-HPLC with UV Detection

A rapid, simple, selective, and specific reverse phase high performance liquid chromatography (RP-HPLC) method with UV detection (315？nm) was developed and validated for estimation of febuxostat from spiked human plasma. The analyte and internal standard (diclofenac) were extracted using LLE with diethyl ether. The chromatographic separation was performed on Shodex C-18-4E (5？μm; ？mm) with a mobile phase comprised of methanol : acetate buffer pH 4, 20？mM (90？:？10？v/v), at a flow rate of 1？mL/min. Febuxostat was well resolved from plasma constituents and internal standard. The calibration curve was linear in the range of 250–8000？ng/mL. The heteroscedasticity was minimized by using weighted least square regression with weighing factor of . The intraday and interday %RSD was less than 15. Results of recovery studies prove the extraction efficiency. Stability data indicated that febuxostat was stable in plasma after three freeze thaw cycles and upon storage at ？20°C for 30 days. 1. Introduction The determination of drugs from biological fluids is essential to pharmacologic and pharmacokinetic studies, which include determinations of the absorption, distribution, metabolism, and elimination of drugs in animals and humans. Biologic fluid assays also support studies of correlations of blood tissue levels of drugs and pharmacologic effects (pharmacodynamics), which are sought during drug development [1–3]. Sample preparation for analysis of biological samples is one of the key factors that determine the extent of recovery of the analyte from the sample matrix. In this context, LLE is the most widely used method for extracting analytes from aqueous biological fluids and separating them from endogenous interferants. LLE also provides a simple means of concentrating the analyte by evaporation of the solvent. The analyte is isolated by portioning between the organic phase and the aqueous phase [4, 5]. Febuxostat is chemically 2-[3-cyano-4-(2-methylpropoxy)phenyl]-4-methylthiazole-5-carboxylic acid (Figure 1). It is a non-purine selective inhibitor of xanthine oxidase that is indicated for use in the treatment of hyperuricemia and gout. Febuxostat inhibits xanthine oxidase noncompetitively, therefore reducing production of uric acid [6]. Several methods are reported in literature for estimation of febuxostat from rat plasma using techniques like LC-MS/MS [7–10], LC-MS [11] and HPLC with fluorescence detection [12–15]. However the methods reported involved use of costly sample preparation tools like solid phase extraction and relied on less commonly available